Limited information is available regarding domains within the epithelial Na+ channel (ENaC) which participate in amiloride binding. We previously utilized the anti-amiloride antibody (BA7.1) as a surrogate amiloride receptor to delineate amine acid residues that contact amiloride, and identified a putative amiloride binding domain WYRFHY (residues 278-283) within the extracellular domain of αrENaC. Mutations were generated to examine the role of this sequence in amiloride binding. Functional analyses of wild type (wt) and mutant αrENaCs were performed by cRNA expression in Xenopus oocytes and by reconstitution into planar lipid bilayers. Wild type αrENaC was inhibited by amiloride with a K(i) of 169 nM. Deletion of the entire WYRFHY tract (αrENaC Δ278-283) resulted in a loss of sensitivity of the channel to submicromolar concentrations of amiloride (K(i) = 26.5 μM). Similar results were obtained when either αrENaC or αrENaC Δ278-283 were co-expressed with wt β- and γrENaC (K(i) values of 155 nM and 22.8 μM, respectively). Moreover, αrENaC H282D was insensitive to sub-micromolar concentrations of amiloride (K(i) = 6.52 μM), whereas αrENaC H282R was inhibited by amiloride with a K(i) of 29 nM. These mutations do not alter ENaC Na+:K+ selectivity nor single-channel conductance. These data suggest that residues within the tract WYRFY participate in amiloride binding. Our results, in conjunction with recent studies demonstrating that mutations within the membrane-spanning domains of αrENaC and mutations preceding the second membrane-spanning domains of α-, β-, and γrENaC alters amiloride's K(i), suggest that selected regions of the extracellular loop of αrENaC may be in close proximity to residues within the channel pore.